The structural basis of β2 integrin intra-cellular multi-protein complexes

Abstract

In multicellular organisms, cell adhesion is a pivotal physiological process which is essential for cell–cell communications, cell migration, and interactions with extracellular matrix. Integrins, a family of large hetero-dimeric type I membrane proteins, are known for driving cell adhesion functions. Among 24 different integrins, four β2 integrins, αL β2, αM β2, αX β2 and αD β2, are specific for cell adhesion and migration of leukocytes. Many cytosolic proteins interact with short cytosolic tails (CTs) of β2 and other integrins which are essential in bi-directional signaling processes. Further, phosphorylation of CTs of integrins regulates binding of intra-cellular proteins and signaling systems. In this review, recent advances in structures and interactions of multi-protein complexes of integrin tails, with a focus on β2 integrin, and cytosolic proteins are discussed along with a proposed future direction.

Introduction

Integrins are hetero-dimeric (α and β subunits) single-domain transmembrane (TM) large proteins which are found throughout the animal kingdom and in metazoans (Hynes 2002; Arnaout et al. 2005). Integrins are essential in many cellular processes like cell adhesion, migration, and proliferation (Hynes 2002; Arnaout et al. 2005). Integrins also participate in the pathogenesis of a number of diseases including cancer, heart disease, blood disorders, and infections (Bachmann et al. 2019; Nolte and Margadart 2020). Therefore, discovering drugs that can target integrins is an active area of research and development (Slack et al. 2022; Cox 2020). Thus far, twenty-four hetero-dimeric integrins are known to exist as combinations among eight β-subunits and eighteen α-subunits. Each of the α- and β-subunits of integrin can be divided, based on cellular localization, into three domain regions: a large extra-cellular domain at the N-terminus, a middle TM domain passing the membrane bilayer, and a relatively short cytoplasmic tail (CT) at the C-terminus (Hynes 2002; Arnaout et al. 2005). The striking functional feature of integrins is that they can transduce cell signals in a bi-directional fashion, termed as “inside-out” and “outside-in.” The inside-out integrin signaling arises from an agonist stimulation of non-integrin receptors, e.g., GPCR, T cell, or chemokine receptors, activating integrin to transduce signals outside of the cells. On the other hand, outside-in integrin signaling is typified by binding of matrix ligands to the extra cellular domain resulting downstream signaling into the cells. The inside-out signaling mechanism of integrin is largely understood from studies of leucocytes and platelet model systems (Gahmberg et al. 2019; Tan 2012; Springer and Dustin 2012; Das et al. 2014). Platelets have also been investigated for the outside-in signaling process. The bi-directional signaling can promote clustering of several integrin molecules across the cell membrane. The clustered integrins have higher affinity and avidity due to the multivalent interactions of the cognate ligands leading to increased cell adhesion and regulation of cellular processes. Atomic resolution structures and interactions of integrins and protein complexes are essential in understanding of the regulatory and allosteric mechanisms involved in bi-directional signaling (Luo et al. 2007; Anthis and Campbell 2011). However, an atomic-resolution structure of any full-length integrin has yet to be solved. Nevertheless, structures have been determined for independent domains of some integrins largely by x-ray and NMR methods. Structures of the large extracellular domains of αV β3, αIIb β3, and αX β2 integrins, either in free or in complex with ligands and antibodies, were determined by x-ray crystallography (Xiong et al. 2001; Zhu et al. 2009; Xie et al. 2010; Xiao et al. 2004). Comparison of negative strain EM images of the extracellular domain of integrins indicated shape changes or global conformational rearrangements occurred upon ligand binding. The structures of the TM domains of αIIb β3 and αL β2 integrins have been obtained by use of solution NMR methods under membrane mimicking conditions (Yang et al. 2009; Lau et al. 2009; Surya et al. 2013). NMR structures and interactions of the CTs of β2 and αIIb β3 integrins have been reported (Qin et al. 2004; Vinogradova et al. 2002; Bhunia et al. 2009; Chua et al. 2011; Chua et al. 2012). Structural studies of the integrin domains along with biochemical and cell-based functional analyses have suggested models of activation and allosteric regulation of the full-length receptors (Hynes 2002; Arnaout et al. 2005). It is understood that the most integrins may undergo three major conformational states in transducing bi-directional signaling (Fig. 1).

Fig. 1.

Models of conformational changes of integrins. A Inter-subunit interactions stabilized bent conformation at the resting state (left). Active states of integrins represented by straight conformations (middle and right). B Switch blade or flick knife and angle poised models differ on TM and CT orientations. Blue: α subunit from extracellular N-terminus, β-propeller; light blue: thigh and calf (1 and 2) domains. Purple: TM (thick straight line), tight blue: cytosolic tail (ragged line). Pink: β subunit from extracellular N-terminus, βIA; yellow: hybrid domain, C-terminus helix; green: PSI domain; purple: I-EGF domain; yellow stick: disulfide bond between the first I-EGF and PSI; purple: β-TD (small circle), TM (thick straight line); green: cytosolic tail (jagged line); star: activation epitopes in I-EGFs. Inward and outward arrows: interactions and separation between the subunits, respectively; lightening arrows: signaling events. Light grey: membrane (thin rectangle). This figure has been reproduced from Hynes, R.O. (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110, 673–687 with permission from Cell Press

In the resting integrin, the closed headpiece assumes a bent conformation that has a low affinity for the activating ligands. In the low affinity state, the extracellular domains of the hetero-dimeric integrin face toward the membrane bilayer and are largely stabilized by intimate interactions between the α and β subunits (Gahmberg et al. 2009; Shimaoka et al. 2002; Xiong et al. 2001). Further, in the resting state, both the TMs and CTs of α and β subunits of integrins can be engaged in non-covalent interactions. Integrins experience extensive rearrangements of the α,β subunits during outside-in or inside-out activation processes (Xiong et al. 2003; Nishida et al. 2006; Takagi et al. 2002; Kim et al. 2011). Two plausible conformations of activated integrins are described, i.e., intermediate affinity and high affinity. These conformations are characterized by global repositioning of the domains of the α,β hetero-dimer stabilizing straighten or extended states of the receptors. The high affinity state of integrin is the most extended with open headpiece that can engage in binding with extracellular matrix ligands, whereas an intermediate affinity state, described in an activated αLβ2 or LFA-1 integrin, is represented as extended conformation but with closed headpiece (Stanley et al. 2008; Li et al. 2007; Tan 2012). The extended conformations of activated integrins are appeared to be maintained by separation of the leg and diminished interactions between the extracellular domains of α and β subunits. The extended integrins also feature disruption of TM/TM packing and separation of cytosolic tails (CTs) interactions. Both the α and β CTs are key elements in bi-directional signal transduction of integrins. Astonishingly, a plethora of cytoplasmic proteins are known to bind to the relatively short CTs which is deemed essential for functional regulation of the receptor (Legate and Fässler 2009; Gahmberg and Grönholm 2022). The sequence of binding events, mutual exclusiveness, and synergistic interplay of multiprotein complexes are among the central mechanisms in integrin research.

Cytosolic tails, phosphorylation, and protein binding

Integrins are bestowed with relatively short CTs (except for β4 CT) which are critically important for the regulation of bi-directional signaling processes (Legate and Fässler 2009; Gahmberg and Grönholm 2022). The primary structures of all the β CTs, except β4 and β8, are similar containing conserved sequence motifs NPxY/F and NxxY/F at the membrane proximal (MP) and membrane distal (MD) region, respectively (Fig. 2). By contrast, α CTs display more variation in amino acid sequences. Although a conserved MP motif GFFKR is shared by most of the members (Fig. 3). Further, selected residues (Thr, Ser, and Tyr) at the canonical motifs of CTs are known to undergo phosphorylation (Fig. 2).

Fig. 2.

The primary structure of β CTs of integrins. Residues of β CTs are numbered according to Tan 2012. The MP and MD conserved motifs, NPxY/F and NxxY/F, respectively, are highlighted in red. Residues known to be phosphorylated are in green. Note that β4 and β8 CT do not contain canonical MP or MD motifs. The β4 integrin has a long CT, entire sequence is not shown here

Fig. 3.

The primary structure of α CTs of integrins. The MP conserved motif GFFKR is highlighted in red, and phosphorytable residues are in green

The inside-out signaling events of integrins including β2, β3, β1, and β7 are manifested by binding of CTs with number of intracellular proteins. In this regard, the β CTs are known to serve as a binding hub of the regulatory intracellular protein complexes (Fig. 4). By contrast, a limited set of proteins are thus far identified to interact with αCTs of integrins. CTs associated proteins can be broadly classified either as activators or repressors. Notably, FERM domain proteins talins-1,2 and kindlins-1,2,3 and scaffold protein 14–3-3ζ are bonafide activators, whereas cytoskeletal protein filamin and adaptor protein Dok-1 are well-studied examples of repressors or negative regulators. Further, protein kinase mediated phosphorylation events of CTs at the conserved sequence motifs are critical in either activation or repression of integrins (Figs. 2 and 3). An interesting interplay of multiprotein complexes occurs among positive and negative regulatory proteins in the context of non-phosphorylated and phosphorylated β2 CT (Chatterjee et al. 2018a, b). Filamin A, a large cytoskeleton protein, acts as a negative regulator of integrins (Sharma, et al. 1995; Calderwood et al. 2001; Kiema et al. 2006; Liu et al. 2000). The domain 21 of filamin or FLNa-Ig21 binds to the MD sequence of non-phosphorylated β2 CT in the resting state of integrin (Calderwood et al. 2001; Kiema et al. 2006; Liu et al. 2000; Das et al. 2011). Association of FLNa-Ig21 with β2 CT is known to further inhibit binding of activator talin and co-activator kindlins (Kiema et al. 2006). Phosphorylation of residue T⁷⁵⁸ at the T⁷⁵⁸TT motif of β2 (also β7) significantly reduces filamin binding while promoted a high-affinity binding of the dimeric 14–3-3ζ protein (Takala et al. 2008). By contrast to filamin mediated negative regulation of non-phosphorylated β CTs, the adaptor protein Dok-1 is known to bind to phosphorylated β3 CT and β2 CT imparting negative regulations of integrins (Calderwood et al. 2003; Oxley et al. 2008; Anthis et al. 2009; Gupta et al. 2015; Niki et al. 2016). Phosphorylation of residue Y⁷⁷³ at the MP NPxY motif in β3 CT enables a high affinity binding of the PTB (phosphotyrosine binding) domain of protein Dok-1 competing out talin binding (Oxley et al. 2008; Anthis et al. 2009). Interestingly, Dok-1 binding to β2 CT follows a non-canonical mode of binding due to the non-phosphorylatable NPxF motif (Fig. 2). Phosphorylation of residue S756, adjacent to NPxF motif, promotes Dok-1 binding to β2 CT (Gupta et al. 2015). Fewer proteins are thus far identified that bind to the α CTs of integrins (Gahmberg and Grönholm 2022). Sharpin is known to bind GFFKR motif of α CTs that could negatively regulate activation of integrins. Recently, binding of filamin domain FLNa-21 to αM CT is reported that appears to play an essential role in the activation of αMβ2 integrin (vide infra).

Fig. 4.

Protein binding sites of the β CT of β2 integrin. β2 CT interacts with various cytosolic proteins employing MP and MD regions. The NPxF motifs are in red, and residues that can be phosphorylated are in green

Structure of integrin β2 cytosolic tails and complexes

Conformations of β2, αL, αM, and αX CTs and CT/CT complexes are characterized by NMR methods (Bhunia et al. 2009; Chua et al. 2011; Chua et al. 2012). Overall, CTs of β2 integrins are largely flexible; however, population of folded conformations could be detected. Only the N-terminal MP part of 46 residue long β2 CT assumes a α-helical structure, and the rest of the β2 CT appeared to be largely disordered (Bhunia et al. 2009). NMR structure of 58-residue long αL CT revealed folded conformations consisted of mutually interacting three helices (Bhunia et al. 2009), although ¹⁵ N relaxation experiments indicated the helical fold of αL CT is dynamically flexible, presumably interconverting with unfolded states. ¹⁵ N-¹H HSQC NMR analyses had revealed transient interactions between αL and β2 CTs (Bhunia et al. 2009). A docked complex of αL/β2 CTs, obtained solely from chemical shift changes, demarcated potential packing among MP helices of β2 and αL CTs (Fig. 5A).

Fig. 5.

Complexes of αL/β2 CTs and αIIbβ3 TMs. A NMR-based docked model of the complex of αL and β2 CTs, in two different orientations. Three helices of 57-residue long αL CT are represented in red, blue, and purple colors; the MP helix of β2 CT is in orange. The interface of the CT/CT complex delineates salt bridges/ionic interactions among helical residues of αL and MP helix of β2 CTs. This figure has been reproduced from Bhunia et al. J Biol Chem. 2009 284(6):3873–84 under the terms of the Creative Commons CC-BY license. NMR-derived structures of TMs of platelet αIIbβ3 integrin B in lipid bicelles and C in organic solvent acetonitrile. Sidechain of residues involved in packings are highlighted in sticks

Atomic resolution structures of N-termini myristoylated αM and αX CTs were solved in membrane mimic detergent micelle solution (Chua et al. 2011; Chua et al. 2012). Myristoylation, as a TM surrogate, has resulted a dramatic structural stabilization of αM, αX, and CTs presumably due to the micelle insertion. Similar observation was made for the αIIβ CT of β3 platelet integrin (Vinogradova et al. 2000). It is noteworthy that seminal studies that were carried out for αIIbβ3 system revealed structures and heterodimerization of the two CTs (Vinogradova et al. 2002). Molecular complex between α and β CTs are critical in maintaining the resting state of integrin. In particular, salt bridge interactions involving residue Arg in the conserved GFFKR motif of α CTs with residue Asp 731 (β2 integrin) are found to be required for the resting state structure (Fig. 5A). α and β CT interactions are among the key elements for integrins bi-directional signaling mechanism. Although atomic resolution structures and interactions of α and β CTs are yet to be determined along with the TM helices embedded in membrane, 3-D structures of αIIbβ3 TMs are reported in membrane like environments using constructs that were lacking the CTs (Fig. 5B, C). It is highly likely that novel structural and dynamical information and interactions could be achieved, while CTs are covalently attached to the respective TMs.

Structures and interactions of binary complexes of β2 cytosolic tails and activators

The β CT of integrins contain consensus motifs that apparently provide specific sites for interactions with an expanding list of intra-cellular proteins. Key signaling activities of integrins, e.g., repression, activation, clustering, and assembly of multi-protein complexes, can be regulated by cytosolic proteins/CTs binding. Despite eminent significance, in-depth analyses of CTs/protein complexes are largely deficient. Here, binary complexes of β2 CT with talin, kindlins, and 14–3-3ζ are discussed and compared with other integrins. Talins 1,2 and kindlins 1,2,3 are recognized as activators and coactivators, respectively, of integrins (Sun et al. 2019; Gough and Goult 2018; Klapholz and Brown 2017; Rognoni et al. 2016; Moser et al. 2009a, b). Dimeric 14–3-3ζ binds to phosphorylated TTT motif of β2 integrin during activation of leucocytes (Takala et al. 2008). Between two isoforms, talin-1, hence referred as talin, is more investigated in terms of structural, biophysical, and functional studies. Talin is a large, 270 kDa, multidomain protein consisted of a head domain, 50 kDa, a long rod domain of 13 helical bundles (R1-R13) and a dimerization (DD) domain (Sun et al. 2019, Gough and Goult 2018; Klapholz and Brown 2017). The talin head belongs to the family of FERM domain protein which is characterized by the presence of four independently folded subdomains F0, F1, F2, and F3 (Roberts and Critchley 2009). Binding of the F3 subdomain of talin head to β CTs of integrins is the leading element in the inside out activation of several integrins (Tadokoro et al. 2003; Nieswandt et al. 2007; Gingras et al. 2009; Anthis and Campbell 2011). Talin assumes a compact autoinhibited state, whereby ligand binding sites are largely masked by “head-rod” interactions (Anthis and Campbell 2011; Campbell and Ginsberg 2004). Whilst head and rod domains are spatially separated in the active state of talin that unmasks binding site for integrin and other proteins, the head domain of talin has garnered most of the attention to realize mechanistic basis of integrin activation. Structural studies of talin head delineated an extended conformation that is not typical of the clover leaf topology of known FERM domains (Elliott et al. 2010; Goult et al. 2013; Dedden et al. 2019). Interestingly, a recent study utilizes so-called conformational stabilization strategy obtaining the canonical FERM domain structure of talin head (Zhang et al. 2020). The membrane proximal NPxY motif of β CTs of integrins is determined to be critical for the binding with F3 sub-domain of talin head. Although atomic-resolution structure of talin with full-length β CT remains elusive to-date, apparently, talin/β CT interactions are transient in nature precluding high-resolution structure determination of the complex (Anthis et al. 2010; Garcia-Alvarez et al. 2003; Wegener et al. 2007). Surprisingly, talin 2 and β1D CT demonstrated a rather higher affinity binding with an estimated K_d of 36 μM and a crystal structure of talin 2 F2F3 in complex of β1D CT could be obtained (Wegener et al. 2007) (Fig. 6A). The structure confirms folding of the MP helix of β1D CT and critical interfacial interactions of the MP NPxY motif with F3 sub-domain of talin head. Notably, the last 13 residues of β1D CT appeared to lack interactions with F2F3 sub-domains of talin and could not be detected within the complex.

Fig. 6.

Structures of complexes of β CTs and activating proteins. A Structure of integrin β1D CT (red ribbon) complex with talin2 F2F3 domain (green ribbon). B Overall topology of the complex of 14–3-3ζ dimer and T758 phosphorylated (pT758) β2 CT peptide (below), ionic interactions at the binding pocket involving sidechains of residue pT758 (red) and K755 with residues of 14–3-3ζ. C (top) Overall fold of the structure of kindlin 2 F3 with β1 CT derived peptide (red), (bottom) residues involved in packing at the interface of the complex, β1 CT (red), and kindlin 2 F3 (green)

Interactions of talin F3 with β2 CT and phosphorylated, at T⁷⁵⁸TT, β2 CT were analyzed by NMR and ITC studies (Chatterjee et al. 2016). Non-phosphorylated and T⁷⁵⁸ phosphorylated β2 CTs demonstrate different binding affinity to talin F3 sub-domain. The K_d values of the complexes were estimated to be 244 μM vs 578 μM, for non-phosphorylated and phosphorylated β2 CTs, respectively. Determination of atomic resolution structure of the binary complex of talin and β2 CT could not be achieved because of the transient nature of binding. Dimeric 14–3-3ζ is a positive regulator of β2 integrins which is known to activate actin cytoskeleton modulators of T cells. Phosphorylation of residue T758 at the TTT motif of β2 CT confers high affinity binding, K_d 4.9 μM, to 14–3-3ζ protein (Takala et al. 2008). A crystal structure of pT758 β2 CT peptide fragment, K⁷⁵⁵SApTTTVM, in complex with 14–3-3ζ revealed canonical mode of interactions within the ligand binding groove (Fig. 6B). Further, kindlin family of proteins, kindlin-1, kindlin-2, and kindlin-3, are recognized as coactivators of integrins (Bu et al. 2021; Plow and Qin 2019). Kindlin-2, expressed in all tissues, activates β1 and β3 integrins but lacks activity for β2 integrin. On the other hand, kindlin-3, expressed in hematopoietic cells, is able to activate several integrins including β2 (Bu et al. 2021; Plow and Qin 2019). Like talin head, the FERM domain of kindlin consists of F1, F2, and F3 subdomains with an additional N-terminus F0 subdomain (Bu et al. 2021; Plow and Qin 2019). The striking feature of kindlin FERM domain is the presence of splits F1 and F2 sub domains. A long loop rich in cationic residues separates the F1 subdomain into two-halves, whereas a folded PH domain is inserted into F2 subdomain. Initial studies have determined the structure of isolated domains F0 and PH and cationic F1 loops of kindlins (Perera et al. 2011; Liu et al. 2011; Goult et al. 2009; Chua et al. 2016; Ni et al. 2017). Recently, atomic resolution structures of kindlin-2 and the full-length kindlin-3 are solved (Bu et al. 2020; Li et al. 2017). These structures of kindlins revealed FERM domain topology, spatial orientation of the PH domain of kindlin-3, and oligomerizations. Kindlin-mediated activation of integrins requires binding of the β CT membrane distal NPxY/F motif with the F3 subdomain (Ma et al. 2008; Larjava et al. 2008). Functional studies have demonstrated leucocyte adhesions, and cell spreading is critically dependent on the activation of β2 integrins (Wen et al. 2022; Moser et al. 2009a, b; Moser et al. 2008). Further, membrane distal N⁷⁶³PKF and T⁷⁵⁸TT motifs of β2 CT provide binding interactions with kindlin-3 (Morrison et al. 2013; Bu et al. 2021). It is pertinent to note that phosphorylation of the first Thr in the TT motif of β1 CT abolishes kindlin-2 binding (Harburger et al. 2009; Böttcher et al. 2022). A plausible ternary complex may occur in the presence of talin binding where kindlin-2 is able to interact with β1 CT with the membrane distal NPxY binding motif. At present, it remains unclear whether similar scenario is present in the regulation of β2 integrin. Phosphorylation of residue T758 at TTT motif of β2 CT raises an interesting possibility of simultaneous binding of talin, 14–3-3ζ, and kindlin-3. Indeed, a ternary complex of talin F3, 14–3-3 ζ, and pT758 β2 CT has been characterized (vide infra).

Kindlins are leading regulatory proteins essential for activation of several integrins, despite atomic resolution structures and interactions of kindlins with β CTs are currently lacking. NMR and biophysical analyses demonstrated that talin head domain and kindlin-2 can bind together without any apparent synergism to β1 and β3 CTs (Bledzka et al. 2012). Recently, structure of kindlin-2 is determined in complex with a β1 CT derived peptide fragment (TTVVNPKY) containing the canonical binding motif (Li et al. 2017) (Fig. 6C). The β1 CT peptide was covalently linked with a truncated variant of kindlin-2 to prevent dissociation from the complex. Regardless, the complex structure has delineated some of the critical interactions between kindlin-2 and β1 CT.

Structures and interactions of binary complexes of β2 cytosolic tails and repressors

Filamin A and Dok-1 are better characterized as the negative regulator of integrins. Both proteins bind to β CTs of integrins and compete with talin binding. Another cytosolic protein named Sharpin has been identified to inhibit β1 integrin activation (Rantala et al. 2011; Kasirer-Friede et al. 2019). Sharpin appears to interact with the α CT of β1 integrin, however, structural details of binding are yet to be determined (Rantala et al. 2011). Among 24 immunoglobulin-like repeats of filamin A, domain 21 or FLNa-Ig21 interacts with β CTs of integrins. Atomic-resolution structures of FLNa-Ig21 in complex with β2 and β7 CTs derived peptides were determined (Kiema et al. 2006; Takala et al. 2008) (Fig. 7). NMR and crystallographic structures of β2 CT MD peptide (P⁷⁵²LFKSATTTVMN⁷⁶³) delineated a stable β-strand conformation of residues S756-V761 in complex with FLNa-Ig21 (Takala et al. 2008; Chatterjee et al. 2018a, b). Typically, the β-strand of β2 CT runs antiparallel with the β-strand C of FLNa-Ig21 and forms multiple backbone-backbone hydrogen bonds. NMR studies further demonstrated dynamic nature of the T⁷⁵⁸TT motif of the β2 CT in the complex that may render a facile phosphorylation of Thr 758 residue (Chatterjee et al. 2018a, b).

Fig. 7.

Structures of complexes of β CTs and repressor protein filamin A. (left) NMR structure of the complex of FLNa-Ig21/β2 CT showing interfacial interactions and β-sheet structure of β2 CT at the binding pocket of β_c and β_d strands of FLNa-Ig21. (middle) Global fold of the x-ray-derived structures of FLNa-Ig21/β2 CT and (right) FLNa-Ig21/β7 CT

Notably, phosphorylated β2 CT at residue Thr 758 reduces filamin binding while enables recruitment of 14–3-3ζ by high affinity interactions during integrin activation. The MD β CTs/ FLNa-Ig21 structures may not fully explain inactivation mechanism of integrins by filamin as talin binding is still feasible to the MP region of β CTs. Recent NMR structures of ternary complexes of FLNa-Ig21 with αM/β2 CTs and αIIb/β3 CTs have provided novel insights toward filamin mediated inhibition of integrin (vide infra).

In cell-based assays, PTB domain of protein Dok-1 was observed to be associated with β CTs of several integrins including β3 and β2 (Calderwood et al. 2003). As shown for platelet αIIbβ3 integrin, Dok-1-mediated negative regulation of integrin requires phosphorylation of residue Y747 at the MP NPxY motif (Oxley et al. 2008; Anthis et al. 2009). The PTB domain of Dok-1 confers a high affinity binding to pY747 β3 CT with an estimated K_d of 37 μM that can potentially compete out talin binding (Fig. 8A) (Oxley et al. 2008). On the other hand, talin displays a higher binding affinity to non-phosphorylated β3 CT. Generally, the PTB domain of Dok-1 binds to phosphorylated Tyr residues in peptide ligands. However, the nonphsophorytable NPxF⁷⁵⁴ motif of β2 CT is unable to permit Dok-1 binding. Residue Ser756 adjacent to NPxF motif has been detected to be phosphorylated (Fagerholm et al. 2002).

Fig. 8.

Complexes of β CTs and repressor protein Dok-1. A A docked model of the complex of pY747β3 CT peptide and Dok-1 PTB domain. Ionic interactions involved residues R207, R221, and R222 of dok-1 with phosphorylated residue pY747 of β3 CT. B and C A docked model of the complex of pS756β2 CT peptide and Dok-1 PTB domain. B Ionic interactions involved residues R207, R221, and R222 of dok-1 with phosphorylated residue pS756 of β2 CT. C Non-polar packing interactions at the interface of pS756β2 CT peptide and Dok-1 PTB domain

Interestingly, Ser756 is phosphorylated β2 CT demonstrated binding to Dok-1 PTB domain with an estimated K_d of 98 μM (Gupta et al. 2015). By contrast, compared to Dok-1, talin binds to non-phosphorylated β2 CT with higher affinity (Chatterjee et al. 2016). Atomic resolution structure of Ser or Tyr phosphorylated β CTs in complex with Dok-1 PTB is yet to be determined. Only a model, based on NMR data, of Ser756 phosphorylated β2 CT peptide in complex of Dok-1 PTB has been reported (Fig. 8B, C). The “phosphor switch” regulating Dok-1 vs talin binding to β CTs has been thought to be contributing to the activation and repression mechanisms of integrins.

Structures and interactions of multiprotein complexes of β2 cytosolic tails

The β CTs possess an extraordinary ability to interact with multiple binding proteins that may essentially involved in allosteric regulations of integrins. Thus, defining structural requirement of multiprotein complexes of β CTs is highly needed. In this section, recent progresses made in determining ternary complexes of β2 and β3 CTs with talin, filamin, 14–3-3ζ, and Dok-1 are discussed.

A ternary complex of talin F3 and 14–3-3ζ and phosphorylated Thr758 β2 CT has been defined by NMR, ITC, and docking studies (Chatterjee et al. 2016). Binary complexes of unmodified β2 CT and phosphorylated Thr758 β2 CT of F3 PTB domain of talin head and 14–3-3ζ, respectively, are characterized (Chatterjee et al. 2016). Notably, binding affinity of talin F3 significantly varied between unphosphorylated (K_d ~ 244 μM) and phosphorylated Thr758 β2 CTs (K_d ~ 578 μM). The lower binding affinity was ascribed to proximity of Thr758 of β2 CT to an acidic residue, D372, of talin F3 (Fig. 9A), while talin F3 and 14–3-3ζ together demonstrated a high affinity complex, K_d 161 μM, with phosphorylated Thr⁷⁵⁸ β2 CT. Comprehensive NMR, ITC and mutagenesis studies revealed structural and dynamical characteristics of a stable ternary complex of talin/14–3-3ζ /phosphorylated β2 CT (Fig. 9B). In the docked complex, phosphorylated β2 CT can bind simultaneously to talin F3 and 14–3-3ζ.

Fig. 9.

Multiprotein complexes of β CTs and cytosolic proteins. A A docked complex of F3 talin and pT758 β2 CT. Note that sidechains of residue D372 of talin F3 and pT758 β2 CT are in proximity. B (top) Topology of NMR-based docked ternary complex of 14–3-3ζ (green ribbon, one subunit of the dimer), talin F3 (magenta), and pT758 β2 CT (red). (bottom) An expanded view of the interactions between pT758 of β2 CT with Arg residues of 14–3-3ζ at the binding pocket. C Topology of NMR-based docked complex of 14–3-3ζ (green ribbon), Dok-1 PTB (cyan), pT758 β2 CT peptide (orange, space-filled), and pY747 β3 CT peptide (red, space-filled). Figure 8C has been reproduced from Chatterjee et al. (2018) Interaction analyses of 14–3-3ζ, Dok1, and phosphorylated integrin β. Cytoplasmic tails reveal a bi-molecular switch in integrin regulation. J. Mol. Biol. 430, 4419–4430 with permission from Elsevier

The MP helix of β2 CT which includes NPxF motif constitutes binding site for talin F3, whereas pT⁷⁵⁸TT in the MD part is engaged within the binding pockets of dimeric 14–3-3ζ. The synergistic engagement of both talin and 14–3-3ζ with β2 CT is likely to promote inside-out and outside-in activation of integrins (Chatterjee et al. 2016). Switching of integrin from the off to on-state requires dissociation of repressor proteins from CTs followed by recruitment of the activating partners. Filamin displacement from β2 CT has been thought to occur as a consequence of phosphorylation of residue T758 at the TTT motif (Takala et al. 2008). This phosphor switch facilitates high-affinity binding to 14–3-3ζ. Filamin can also be displaced from β CT upon binding with LIM domain of migfilin that may facilitate activation of integrins (Das et al. 2011). By contrast, binding of the negative regulator Dok-1 to integrins is promoted by phosphorylation either at residues Y⁷⁷³ or S⁷⁵⁶ of β3 or β2 CTs, respectively (Oxley et al. 2008; Gupta et al. 2015). Interactions between positive and negative regulatory proteins might be relevant to the fine tuning of activation of integrins. A novel bi-molecular switch has been deduced in a ternary interplay among 14–3-3ζ, Dok-1 PTB, and β2 and β3 CTs (Chatterjee et al. 2018a, b). The dimeric scaffold protein 14–3-3ζ demonstrates discernable complex formation with the PTB domain of Dok-1. Interestingly, 14–3-3ζ/Dok-1 PTB complex is able to recognize specific binding to phosphorylated β2 and β3 CTs (Fig. 9C). In particular, such a protein complex consisted of positive and negative regulators could be essential in sustaining the rapid kinetics of integrins bi-directional signaling (Chatterjee et al. 2018a, b).

Mechanism of filamin A mediated negative regulation of integrins is largely understood from structures and interactions with the C-terminal MD region of β CTs. Recent studies demonstrates that the N-terminal MP helix also interacts with filamin FLNa-Ig21in full-length β2 and β3 CTs (Zhiping et al. 2021, Liu et al. 2015). Atomic resolution structure of platelet αIIbβ3 revealed a novel ternary complex of FLNa-Ig21 with β3 and αIIb CTs (Liu et al. 2015) (Fig. 10A). The β3 and αIIb CTs synergistically bind to FLNa-Ig21 with high affinity.

Fig. 10.

β2 and αM CT complexes with filamin. A NMR-based docked complex of αM CT (red) and FLNa-Ig21 (green). Residue Y1137 of αM CT showed interfacial packings with residues F2285 and L2271 of FLNa-Ig21. B NMR-based docked complex of β2 CT (red) and FLNa-Ig21 (green). Residues I727, L732, and V761 belonging to MP helix of β2 CT are engaged in van der Waals packing interactions with residues A2268; L2271 of FLNa-Ig21 are highlighted. C A docked model of the ternary complex of αM CT (cyan), β2 CT (red), and FLNa-Ig21 (green). In the ternary complex, αM CT MP helix shows most interactions with β2 CT MP helix

The stable ternary complex of FLNa-Ig21/CTs shows an estimated K_d of 19 μM, whereas the binary complexes are of low affinity. In the ternary structure, β3 CT shows an extended binding interface, involving MP helix and MD β-sheet, with FLNa-Ig21, whereas αIIb has only fewer interactions with filamin FLNa-Ig21. Regardless, the mode of engagement of β3 and αIIb CTs with filamin has provided mechanistic insights of inactivation of platelet αIIbβ3 integrin. A more recent structural and functional study has employed β2 and αM CTs of αMβ2 integrin in complexes of filamin (Zhiping et al. 2021). Binary complexes of the full-length CTs of αMβ2 integrin with FLNa-Ig21 are characterized (Fig. 10B). This study has elucidated a novel binary complex between αM subunit of integrin and filamin, whereby the MP helix of αM CT occupies the canonical ligand binding pocket of FLNa-Ig21. The binary complex could be detected in cell-based studies (Zhiping et al. 2021). Notably, interactions between αM CT/filamin FLNa-Ig21 are diminished in a latent or inactivated state of αMβ2 integrin. However, a prevalent binding between filamin FLNa-Ig21 with αM CT could be detected in stimulated cells when αMβ2 integrin is activated. A docked model, based on NMR chemical shift changes, is deduced showing potential interfacial residues of the complex (Fig. 10B). Mutation of the interfacial residue Y1137 to Gly of αM CT has significantly reduced in vivo interactions with filamin (Zhiping et al. 2021). The full-length β2 CT interacted with FLNa-Ig21 domain of filamin employing residues of MP helix and MD β-strand. A model of the ternary complex of FLNa-Ig21 with α and β CTs of αMβ2 is derived based on HADDOCK (Fig. 10C). The αMβ2/FLNa-Ig21 structure closely resembles that of atomic resolution structure of platelet αIIbβ3/FLNa-Ig21 defining a common mode of inhibition of β2 and β3 integrins by filamin. Although, it is noteworthy that in the ternary complex filamin binds to αIIbβ3 CTs with a remarkably higher affinity in comparison to that of αMβ2 CTs. In fact, weak interactions are predominantly observed in cytosolic protein complexes regulating β2 integrins. Presumably, transient interactions of β2 integrins could be important for leucocyte functions which are distinctly different from the platelet.

Concluding remarks and future perspectives

The bi-directional signaling of integrins is prevalently controlled by the interactions of cytosolic proteins and CTs. Astoundingly, a large number of cytosolic proteins are capable of interacting with the β CTs of integrins. Regulation of integrins can involve formation of muti-protein complexes within β CTs. Understanding integrin biology thus requires in-depth analyses, and atomic-resolution structures of the multi-protein complexes. However, transient complexes of CTs with most regulatory proteins may complicate high-resolution structure determination. The TM-helices of heterodimeric integrins connect the large extracellular domains with CTs and undergo substantial structural changes in the signaling process. However, structures and protein interactions of CTs have mostly been investigated in the absence of TM domains. Investigations of protein binding and structures of CTs could be of significance interest, while they are attached to the TM helices. Indeed, stable 3-D structures of membrane inserted acylated CTs of β2 and β3 integrins are obtained (Vinogradova et al. 2000; Chua et al. 2011; Chua et al. 2012). Interaction affinity between αM and β2 CTs is enhanced when CTs are in membrane-like environments. Thus, development of novel methods and sample preparations are needed for atomic resolution structures of CTs + TMs/protein complexes. Further, separate domains of talin and kindlins are known to interact with membrane which is important in modulating activation of integrins. NMR-based methods could be useful in this regard since TM helices can be inserted in lipid bicelle or nanodiscs. Atomistic structures of CTs + TM/protein complexes will greatly help in resolving mechanisms of bi-directional signaling of integrins.

Funding

Studies of integrins are support by grants from Biomedical Research Council (BMRC), Singapore and Ministry of Education (MOE), Singapore.

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Conflict of interest

The author declares no competing interests.